全 文 ：Received 26 Apr. 2004 Accepted 14 Jun. 2004
Supported by the National Natural Science Foundation of China (30025030), the National Special Program for Research and Industrializa-
tion of Transgenic Plants (J00-A-006-1) and 2000’s Trans-century Talent Development Program of Ministry of Education, China.
* Contributed equally with the first author.
** Author for correspondence. Tel: +86 (0)25 84396029; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (11): 1366-1372
Cloning and Characterization of a Ubiquitin Fusion Degradation
Protein Gene in Wheat
HUANG Xian-Zhong1, 2, WEI Ling-Zhu1*, MA Zheng-Qiang1**
(1. State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China;
2. College of Bioengineering, Shi Hezi University, Shi Hezi 832003, China)
Abstract: Ubiquitin fusion degradation (UFD) protein, encoded by the UFD1 gene that was first described
in yeast, is a key component in this Ub-dependent degradation system or Ub fusion degradation pathway. We
isolated a UFD1 -like gene in wheat (Triticum aestivum L.) through RT-PCR. The entire coding region is 948
bp and encodes a polypeptide of 315 amino acids with 74% homology to a UFD1-like protein of Arabidopsis
deposited in the GenBank. Its N-terminal possesses the so-called UFD1 domain highly conserved among
eukaryotic organisms. This gene was designated as TUFD1 and mapped to group 6 chromosomes of wheat.
Southern hybridization and database search demonstrated that UFD1 genes in plants are in single copy or
low copies. Plant UFD1 proteins have three highly conserved C-terminal domains besides the UFD1 domain
and are highly homologous to one another. TUFD1 showed a constitutive expression pattern in roots,
stems, coleoptiles, and leaves of seedlings, and in young spikes and seeds at the hard-dough ripening stage.
Key words: wheat (Triticum aestivum); ubiquitin fusion degradation protein; TUFD1; gene expression
Johnson e t a l . (1992) d iscovered in yeas t
(Saccharomyces cerevisiae) that natural or fusion proteins
bearing a N-terminal ubiquitin (Ub) moiety are rapidly de-
graded in vivo, because the Ub serves as an autonomous,
primary degradation signal. This Ub-dependent degrada-
tion system is called Ub fusion degradation pathway or
ubiquitin fusion degradation (UFD) pathway. By analyz-
ing mutations that perturb the UFD pathway, they identi-
fied at least five genes involved in this process (Johnson
et al., 1995). Among them, UFD1 and UFD5 function at
post-translation steps, UFD3 controls the concentration
of Ub in the cells, UFD2 and UFD4 may influence the
formation and topology of the multi-Ub chains linked to
the Ub-moiety bearing proteins. Taxis et al. (2003) pre-
sented evidence that UFD1 is also involved in the Ub-
conjugation degradation pathway, in which Ub functions
as the secondary degradation signal of the substrate pro-
teins by covalently linked to them.
Some of the UFD genes have been isolated in other
eukaryotes from Dictyostelium discoideum to mammals,
for example, UFD1 homologs in human (Pizzuti et al., 1997),
mouse (Botta et al., 1997), Gallus gallus, Xenopus laevis
and Drosophila melanogaster (Ratti et al., 2001), and UFD2
in human (Krona et al., 2003) and mouse (Kaneko et al.,
2003). UFD1-like proteins have been predicted from
nucleotide sequences of many species including plants.
The homology across the taxa implies that the UFD path-
way has been conserved in the evolutionary history of
eukaryotic organisms.
Ub-mediated proteolysis is essential for normal growth
and has been documented in diverse biological processes,
such as cell differentiation, stress responses, cell cycle
control, regulation of transcription and programmed cell
death (Hershko and Ciechanover, 1998; Pickart, 2001;
Hemmo et al., 2002). In this report, we presented the isola-
tion and characterization of an UFD1-like gene in wheat.
1 Materials and Methods
1.1 Plant materials
D-Sumai No. 3 and C.S. nulli-tetrasomic lines were used
in this study. D-Sumai No. 3 was developed by backcross-
ing a Rht-B1C donor for 23 generations using Sumai No. 3
as the recurrent parent (Zhao et al., 1986).
1.2 Extraction of total RNA
The germinated seeds of D-Sumai No. 3 in Petri dishes
were treated at 4 ℃ refrigerator for 24 h to ensure the uni-
formity and were then left at room temperature for vegeta-
tive growth. Two weeks later, roots, coleoptiles, stems and
leaves of the seedlings were harvested for RNA extraction.
The young spikes and seeds at hard-dough ripening stage
HUANG Xian-Zhong et al.: Cloning and Characterization of a Ubiquitin Fusion Degradation Protein Gene in Wheat 1367
of D-Sumai No. 3 were also collected for RNA extraction.
RNA was extracted with the Trizol kit (Gibco BRL, USA)
according to the manufacturer’s protocol.
1.3 RT-PCR
To synthesize the first strand cDNA, two µg of total
RNA from D-Sumai No. 3 mixed with 10 pmol oligo (dT)12-
18 primers was treated at 70 ℃ for 10 min and then cooled
on ice for 2 min. After adding 2 µL 10× first strand buffer,
0.5 mmol/L dNTP mix, 10 mmol/L DTT and 200 U Super-
script Ⅱ reverse transcriptase in a 20 µL reaction volume,
the sample was incubated at 42 ℃ for 1 h. RNase H was
used to remove RNA in the DNA-RNA hybrids.
The p r imers used fo r RT-P CR, RDF1 (5 -
TCCCAAAGCCGAAACCG-3) and RDR3 (5 -
ACGACGAGGAGGAGGATGAC-3) were designed accord-
ing to the Rht gene homologs in order to isolate the Rht-
B1c gene. The PCR reaction was performed on an RTC-225
PCR apparatus (MJ Research, USA) in a 25 µL volume
containing 1 µL of the synthesized cDNA, 2.5 µL 10×
PCR buffer, 200 nmol/L of each of the primers, 200 µmol/L
each dNTP, 1.5 mmol/L MgCl, 1 U Taq polymerase. The
amplification profile used was 35 cycles of 94 ℃ 30 s, 62 ℃
45 s, 72 ℃ 1.5 min, and a final extension of 5 min at 72 ℃.
The product was resolved in 1.0% agarose gel. This PCR
resulted in amplification of a 839 bp partial cDNA sequence,
which has an open reading frame encoding a polypeptide
with 83% homology to a putative UFD1 protein of
Arabidopsis and 66% homology to the yeast UFD1 protein.
At the nucleotide level, this sequence has 90% homology
to the rice cDNA clone J033094A11 (accession No.
AK121784 ) and 87% to the maize cDNA clone CL14668_1
(accession No. AY112612). Sequence comparison showed
that this sequence was derived from the false amplifica-
tion caused by the complete homology between the 3 end
sequences (9-10 bp) of the two primers with portions of
the UFD1 sequences in wheat.
To isolate the 3 end sequence of the UFD1 gene, 3
RACE cDNA template was prepared according to the
SMART RACE cDNA amplification kit user manual
(Clontech, USA). PCR was performed in 50 µL volume con-
taining 1 µL of the cDNA, 200 nmol/L each of the primers
(UFD3: 5-TGACTTTGCCCCACCACTTGAC-3 and the
universal primer mix provided in the kit), 5 µL 10× PCR
buffer, 200 nmol/L each dNTP, 1.5 mmol /L MgCl2, 2 U Taq
polymerase. PCR was performed with 5 cycles of 94 ℃ 2
min, 72 ℃ 3 min; 5 cycles of 94 ℃ 30 s, 70 ℃ 45 s, 72 ℃ 3
min; 25 cycles of 94 ℃ 30 s, 68 ℃ 45 s, 72 ℃ 2 min, and
followed by a final extension of 5 min at 72 ℃.
Primers UFD5F (5-GCCGAAACCGAGGACGACTCTT-
CTC-3), locating in the 5 UTR region, and UFD3R (5-
AGCCCTGATGATGTTTGTGACAG-3) locating in the 3
UTR region, were designed to amplify the whole coding
region. The amplification profile was as follows: 94 ℃ 3
min; 32 cycles of 94 ℃ 50 s, 58 ℃ 50 s and 72 ℃ 1.5 min;
then 72 ℃ 5 min for final extension.
1.4 PCR cloning and sequence analysis
PCR products were ligated into the pGEM-T Easy vec-
tor (Promega, USA) using the standard procedure. The
positive clones identified were sequenced in Shengyou,
Shanghai. MacVector 7.0 (Accelrys Inc., USA) was used
for analysis of the open reading frame, translation and
ClustalW alignment. Homology search was conducted
through NCBI BLAST.
1.5 Southern blot
DNA extraction was described by Ma et al. (1995). Fif-
teen µg of DNA, digested by EcoRⅠ, EcoRⅤ, HindⅢ
and DraⅠ, respectively, was run on 0.9% agarose gels
and then transferred to Hybond N+ membrane (Amersham,
USA). The probe was prepared by random hexamer primed
a-32P dCTP labeling. Hybridization was performed at 65 ℃
in hybridization buffer (0.5 mmol/L Na Phosphate, pH 7.2,
7% SDS, 1% BSA, 200 ng/L denatured salmon sperm DNA)
overnight. The filters were then washed using 2× SSC/
0.1% SDS, 1× SSC/0.1% SDS and 0.5× SSC/0.1% SDS at
65 ℃, respectively, each for 15 min.
1.6 Semi-quantitative RT-PCR
Semi-quantitative RT-PCR was conducted using the
gene specific primer UFD3 and UFD3R, and the first strand
cDNA of roots, stems, leaves, coleoptiles, spikes and seeds
of D-Sumai No.3 as the templates. The PCR amplification
profile was as follows: 94 ℃ 3 min, then 22-26 cycles of 94
℃ 40 s, 58 ℃ 40 s and 72 ℃ 1 min, finally 72 ℃ 5 min for
extension. In the PCR reaction mix, primers for histone 3
gene of wheat, H3F (5- CATGGCCCGCACCAAGCAGA-
3) and H3R (5-TTGGCGTGGATGGCGCAGAG-3), were
added to serve as the internal control.
2 Results
2.1 Cloning of the cDNA for ubiquitin-fusion degrada-
tion protein
Using the 3 RACE PCR primer designed according to
the 839 bp partial UFD1 sequence, a 684 bp product was
obtained through 3 RACE-PCR. Its sequence overlaps with
the 839 bp sequence in the 180 bp region to its 3 end with
single nucleotide polymorphism at only four different
positions, and has a 249 bp 3 UTR. Thus, a 1 343 bp cDNA
contig for the UFD1-like gene was constructed according
to the sequence alignment.
Acta Botanica Sinica 植物学报 Vol.46 No.11 20041368
To isolate the full coding DNA sequence (CDS), gene-
specific primers UFD5F and UFD3R were designed against
the 5 and 3 UTR of the contig, respectively. The PCR
produced a 1.1 kb band, from which a 1 135 bp sequence
was obtained. It has a 954 bp ORF and encodes a 35-kD
protein of 317 amino acids (aa) (Fig.1). The CDS has 71%
homology to a Arabidopsis thaliana UFD1 gene
(accession No. AY087347, AUFD1a in Fig.2). Thus, this
UFD1-like gene of wheat was designated as TUFD1. At
the amino acid level, the similarity of TUFD1 to AUFD1a is
71% as a whole and over 84% in the 199-aa N-terminal
region, which encompasses the whole conserved N-termi-
Fig.1. The coding DNA sequence of TUFD1 and the amino acid sequence deduced from it. The GenBank accession number for this
nucleotide sequence is AY587885. *, stop codon.
nal domain of UFD1 proteins. There is also 71% homology
between the conserved N-terminal domains of TUFD1 and
yeast UFD1.
Southern blot with C.S. nulli-tetrasomic lines indicated
that TUFD1 locates in group 6 chromosomes of wheat
(Fig.2). The banding pattern implied that TUFD1 is a single
copy or low copy gene in wheat genome. No polymor-
phism between the PCR products of Sumai No. 3 and D-
Sumai No. 3 genomic DNA, amplified with primers UFD5F
and UFD3R, was observed (data not shown).
2.2 Conservation of UFD1 across plant species
To assess the conservativeness of UFD1 protein among
HUANG Xian-Zhong et al.: Cloning and Characterization of a Ubiquitin Fusion Degradation Protein Gene in Wheat 1369
Fig.2. Southern hybridization of the C.S. nulli-tetrasomic lines
using TUFD1 cDNA as the probe. Only part of the lines were
shown. The arrowheads indicate the missing bands, which are
corresponding to the missing chromosomes in the specific nulli-
tetrasomic lines. N, nulli; T, tetra.
Fig.3. The conserved domains of plant UFD1 proteins. a. the UFD1 domain. b. domains Ⅱ, Ⅲ and Ⅳ. AUFDla (accession No.
AY087347), AUFDlb (accession No. NM_202980) and AUFDlc (accession No. NM_201828), Medicago truncatula gene MUFDl (accession
No., AC140035), rice genes OUFDla (accession No. AK121784) and OUFDlb (accession No. AK073711) were retrieved from GenBank.
←
Acta Botanica Sinica 植物学报 Vol.46 No.11 20041370
plant species, multiple alignments were conducted for
amino acid sequences predicted from TUFD1, Arabidopsis
thaliana genes AUFD1a , AUFD1b and AUFD1c,
Medicago truncatula gene MUFD1, and rice genes
OUFD1a and OUFD1b. The size of these proteins varies
from 312 aa to 320 aa. The comparison revealed four con-
served domains, including the largest N-terminal UFD1
domain (domain Ⅰ) (Fig.3a) and domains Ⅱ, Ⅲ and Ⅳ to
the C-terminal (Fig.3b). On average, the conserved regions
account for over 75% of the whole polypeptide sequences.
2.3 Expression of TUFD1 gene
To examine the expression patterns of TUFD1, quanti-
tative RT-PCR was conducted using RNA from coleoptiles,
roots, stems, leaves of the seedlings, young spikes and
seeds at hard-dough ripening stage (Fig.4). Three sets of
PCR reactions were conducted with 22, 24 and 26 cycles,
respectively. The internal control amplified well in all cases.
For TUFD1, the band intensity was almost the same as
that of H3 with 26 cycles, but was much weaker when am-
plified with 22 or 24 cycles, indicating that the amplifica-
tion was still before the plateau stage with less than 24
cycles. According to the band intensity from 22-cycle am-
plification (Fig.4, the upper panel), TUFD1 expressed con-
stitutively in the tissues investigated and the abundance
of its transcripts was far below that of the H3.
3 Discussion
Ub is a highly conserved 76-amino acid protein present
in all eukaryotes (Vierstra, 1996; Galan and Peter, 1999) .
Natural proteins or fusion proteins with a N-terminal Ub
moiety in yeast degrade rapidly in vivo through the UFD
pathway, where the Ub moiety serves as the primary deg-
radation signal (Johnson et al., 1992). In the Ub-conjuga-
tion degradation pathway, Ub functions as the secondary
degradation signal by conjugating with the target proteins,
Fig.4. RT-PCR of TUFD1 with different numbers of PCR
cycles using UFD3 and UFD3R primers. H3 amplification was
used as the internal control. M, fx174/HaeⅢ, Lanes 1-6, roots,
stems, leaves, coleoptiles, spikes and seeds; lane 7, empty control.
which is a common feature for a large portion of intracellu-
lar eukaryotic protein proteolysis. UFD1 is a key gene as-
sociated with both degradation pathways. Even though
UFD1-like genes have been cloned or predicted in a num-
ber of eukaryotic organisms including mammals and plants
using ESTs or genome DNA sequences, TUFD1 was the
first of plant UFD1 homologs characterized. Its nucleotide
sequence has been deposited in GenBank with the acces-
sion number AY587885.
The low copy nature of TUFD1 in wheat may be true
for other plant species. In rice and Arabidopsis only three
homologs have been identified respectively based on gene
prediction from the whole genome sequences. All the
UFD1-like genes encode 34-40 kD proteins, which have a
highly conserved N-terminal domain. In plants, from mono-
cots to dicots, the conservation is prominent at both nucle-
otide and protein levels. Except for the N-terminal domain,
there are three conserved C-terminal domains among the
plant UFD1-like proteins compared (Fig.3). The high level
of homology implies a strong evolutionary conservation
of the UFD1 proteins, and likely the UFD degradation path-
way in plants and even all the eukaryotic organisms. The
N-terminal domain is known for functioning as a degrada-
tion signal of proteins, but the functions of the conserved
C-terminal domain are poorly understood.
Quantitative RT-PCR showed that TUFD1 is expressed
constitutively in wheat with moderate abundance. In
Drosophila, dUFD1L is expressed through the embryonic,
larval and pupa development, as well as in the adult fly
(Ratti et al., 2001). The human UFD1L gene (HUFD1L)
also displays housekeeping features in its promoter char-
acteristics and expression profiles (Novelli et al., 1998). A
high level of HUFD1L gene expression was observed dur-
ing embryogenesis, especially in the eyes and inner ear
primordial, suggesting its role in the fate determination of
ectoderm-derived structures, including neural crest cells
(Pizzuti et al., 1997). HUFD1L locates at the 22q11.2 chro-
mosome region, whose deletion is associated with devel-
opmental defects such as the DiGeorge and velo-cardio-
facial syndromes (Pizzuti et al., 1997; Yamagishi et al., 1999).
In yeast UFD1 gene is required for viability of vegetative
cells (Johnson et al., 1995). These results reflect the func-
tional importance of UFD1-like genes.
Protein degradation is an integral component of cell
physiology. Currently, little is known for the biochemical
roles of UFD1 proteins in plant growth and development.
Further studies on the expression of TUFD1 in various
growth conditions and its loss-of-function effect will shed
light on this issue.
HUANG Xian-Zhong et al.: Cloning and Characterization of a Ubiquitin Fusion Degradation Protein Gene in Wheat 1371
(Managing editor: ZHAO Li-Hui)
Crop Sci, 35: 1137-1143.
Novelli G, Mari A, Amati F, Colosimo A, Sangiuolo F, Bengala
M, Conti E, Ratti A, Bordoni R, Pizzuti A, Baldini A, Crinelli
R, Pandolfi F, Magnani M, Dallapiccola B. 1998. Structure
and expression of the human ubiquitin fusion-degradation gene
(UFD1L). Biochim Biophys Acta, 1396: 158-162.
Pickart C M. 2001. Mechanisms underlying ubiquitination. Annu
Rev Biochem, 70: 503-533.
Pizzuti A, Novelli G, Ratti A, Amati F, Mari A, Calabrese G,
Nicolis S, Silani V, Marino B, Scarlato G, Ottolenghi S,
Dallapiccola B. 1997. UDD1L, a developmentally expressed
ubiquitination gene, is deleted in CATCH22 syndrome. Hum
Mol Genet, 6: 259-266.
Ratti A, Amati M, Bozzali E. 2001. Cloning and molecular char-
acterization of three ubiquitin fusion degradation 1 (UFD1)
ortholog genes from Xenopus laevis, Gallus gallus and Droso-
phila melanogaster. Cytogenet Cell Genet, 92: 279-282.
Taxis C, Hitt R, Park S H, Deak P M, Kostova Z, Wolf D H.
2003. Use of modular substrates demonstrates mechanistic
diversity and reveals differences in chaperone requirement of
ERAD. J Biol Chem, 278: 35903-35913.
Vierstra B D. 1996. Proteolysis in plants: mechanisms and
functions. Plant Mol Biol, 32: 275-302.
Yamagishi H, Garg V, Matsulka R, Thomas T, Scivastava D.
1999. A molecular pathway revealing a genetic basis for hu-
man cardiac and craniofacial defects. Science, 283: 1158-1160.
Zhao Y-H, Zou M-L , Ma Z-Q , Wang S . 1986. Utilization of Rht3
gene in hybrid wheat. J Jiangsu Agr Sci , 2: 1-6. (in Chinese
with English abstract)
References:
Botta A, Jurecic V, Pizzuti A, Novelli G, Dallapiccola B, Baldini
A. 1997. Assignment of the gene for an ubiquitin fusion degra-
dation protein (UFD1) to mouse chromosome 16B1-B4,
syntenic with the Tuple1 gene. Cytogenet Cell Genet, 77: 264-
265.
Galan J, Peter M. 1999. Ubiquitin-dependent degradation of
multiple F-box proteins by an autocatalytic mechanism. Proc
Natl Acad Sci USA, 96: 9124-9129.
Hemmo H, Meyer, Wang Y, Warren G. 2002. Direct binding of
ubiquitin conjugates by the mammalian p97 adaptor
complexes, p47 and UFD1-Np14. EMBO J, 21: 5645.
Hershko A, Ciechanover A. 1998. The ubiquitin system. Annu
Rev Biochem, 67: 425-479.
Johnson E S, Bartel B, Seufert W, Varshavsky A. 1992. Ubiquitin
as a degradation signal. EMBO J, 11: 497-505.
Johnson E S, Ma P C, Ota I M, Varshavsky A. 1995. A pro-
teolytic pathway that recognizes ubiquitin as a degradation
signal. J Biol Chem, 270: 17442-17456.
Kaneko C, Hatakeyama S, Matsumoto M, Yada M, Nakayama
K, Nakayama K I. 2003. Characterization of the mouse gene
for the U-box-type ubiquitin ligase UFD2a. Biochem Biophys
Res Commun, 300: 297-304.
Krona C, Ejeskar K, Abel F, Kogner P, Bjelke J, Bjork E, Sjoberg
R M, Martinsson T. 2003. Screening for gene mutations in a
500 kb neuroblastoma tumor suppressor candidate region in
chromosome 1p; mutation and stage-specific expression in
UBE4B/UFD2. Oncogene, 22: 2343-2351.
Ma Z Q, Sorrells M E. 1995. Genetic analysis of fertility restora-
tion in wheat using restriction fragment length polymorphisms.